enzymes. However, many genes in the microbiome remain uncharacterized due to the challenge in culturing intestinal strains in vitro, which limits the identification of target enzymes. To address this issue, we developed an effective Activity-Based MetaProteomics (ABMP) strategy using a specific activity-based probe (ABP) to screen the entire gut microbiome for target enzymes, without the need to isolate and culture bacterial strains individually. α-Galactosidases (AGALs) are widely distributed in gut microbiota, plant, and animal kingdoms of life and have multiple applications in industries such as food, animal feed, and biomedical sectors. Despite the rich source of AGALs in the gut microbiome, many AGAL genes lack functional annotations. Using an activity-based cyclophellitol aziridine probe specific to AGAL, we successfully identified and characterized several new enzymes possessing AGAL activities from the gut microbiome.